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Published in final edited form as:


Neuroreport. 2011 October 26; 22(15): 767772. doi:10.1097/WNR.0b013e32834ae348.

Intersectin 1 contributes to phenotypes in vivo: implications for


Down Syndrome
Michael P. Hunter1, Marianela Nelson2, Michael Kurzer1, Xuerong Wang1, Richard J.
Kryscio4, Elizabeth Head4,5, Graziano Pinna2, and John P. OBryan1,3,*
1Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612
2Department
3Program

of Psychiatry, University of Illinois College of Medicine, Chicago, IL 60612

in Neuroscience, University of Illinois College of Medicine, Chicago, IL 60612

4Sanders-Brown
5Department

Center on Aging, University of Kentucky, Lexington, KY

of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, KY

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Abstract
Intersectin 1 (ITSN1) is a chromosome 21 (HSA21) gene product encoding a multi-domain
scaffold protein that functions in endocytosis, signal transduction and is implicated in Down
Syndrome, Alzheimers Disease, and potentially other neurodegenerative diseases through
activation of c-Jun N-terminal kinase (JNK). We report for the first time that ITSN1 proteins are
elevated in Down Syndrome individuals of varying ages. However, ITSN1 levels decreased in
aged Down Syndrome cases with Alzheimers Disease-like neuropathology. Analysis of a novel
ITSN1 transgenic mouse reveals that ITSN1 overexpression results in a sex-dependent decrease in
locomotor activity. This study reveals a link between overexpression of specific ITSN1 isoforms
and behavioral phenotypes and has implications for human neurodegenerative diseases such as
Down Syndrome and Alzheimers Disease.

Keywords
scaffold protein; MAP kinase; endocytosis; signal transduction; neurodegeneration; SH3 domain;
EH domain

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INTRODUCTION
Down Syndrome is the most common form of chromosomal trisomy in humans with an
incidence of 1 in 733 live births [1]. Triplication of HSA21 leads to a number of
characteristic phenotypes including musculoskeletal defects, mental retardation, heart
defects, and early-onset Alzheimers disease neuropathology [2]. One of the earliest
observed phenotypes of both Down Syndrome and Alzheimers Disease patients are
abnormally large early endosomal compartments, suggestive of defects in receptor
trafficking [3]. Indeed, nerve growth factor transport is impaired in basal forebrain

Address correspondence to: John P. OBryan, Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL
60612: Tel: 312-996-6221; Fax: 312-996-1225; obryanj@uic.edu.
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cholinergic neurons (BFCNs) in a Down Syndrome mouse model leading to degeneration of


these neurons [4].

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ITSN1 is a unique, multi-domain scaffold protein encoded by a gene located at 21q22.1q22.2. ITSN1 mRNA is elevated in Down Syndrome brains [5]. ITSN1 consists of two
major splice products, ITSN1-short (S) and long (L). Both isoforms possess two Eps15
homology domains, a coiled-coil region, and five Src homology 3 domains; however,
ITSN1-L possesses an extended C-terminus encoding a Dbl homology domain, Pleckstrin
homology domain, and a C2 domain. The DH-PH modules function in concert as a guanine
nucleotide exchange factor that specifically activates the Cdc42 GTPase [6].

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Several lines of evidence suggest a link between Down Syndrome neuropathology and
ITSN1 (reviewed in [6]). First, ITSN1 acts as an endocytic scaffold regulating the assembly
of clathrin-coated pits and alterations to its expression may contribute to the observed
endocytic defects in Down Syndrome. Second, ITSN1 overexpression in model systems
results in defective receptor regulation. Third, ITSN1 enhances activation of the JNKMAPK pathway. As a specific initiator of the apoptotic response, JNK has been implicated
in neurodegeneration [7]. Fourth, whole genome expression analyses reveal that ITSN1 is
one of the most highly induced transcripts in Alzheimers Disease brains [810], consistent
with the development of an Alzheimers Disease-like neuropathology in Down Syndrome
patients above the age of 40 years [2]. Finally, ITSN1 overexpression enhances
neurodegeneration in vivo in a Drosophila model for polyglutamine expansion diseases [11].
These effects involve the JNK pathway and support the notion that ITSN1 and JNK mediate
the response of neurons to toxic stress. Together, these data suggest that ITSN1 may
contribute to the sequelae of Down Syndrome.
ITSN1 expression is widely reported to be elevated in Down Syndrome; however, only a
single study has examined this issue, focusing specifically on ITSN1 mRNA levels [5].
Given this paucity of data and lack of information on ITSN1 protein levels in Down
Syndrome individuals, we examined the expression of ITSN1 proteins in normal and Down
Syndrome cases. Our study represents the first analysis of ITSN1 protein levels throughout
the human lifespan. Finally, we describe the initial characterization of a brain-specific
ITSN1-S transgenic mouse which exhibits specific behavioral phenotypes. In conclusion,
our findings indicate that ITSN1 proteins are overexpressed in Down Syndrome brains and
may contribute to altered behavior in vivo.

MATERIALS AND METHODS


Subjects

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Frozen tissue blocks from the midfrontal cortex were obtained from a total of 25 individuals
(15M, 10F) with Down Syndrome ranging in age from 5 months to 62 years of age. A series
of 16 control cases (8M, 8F) ranging in age from 6 months to 67 years were used for
comparison. Down Syndrome and young or non-demented aged control cases were obtained
from two sources: (1) the UCI-ADRC Brain tissue repository and; (2) NICHD Brain and
Tissue Bank for Developmental Disorders.
Tissue isolation
ITSN1-S transgenic mice were generated as described in Supplemental Digital Content,
Supplemental Digital Content 1, http://links.lww.com/WNR/A147. Mice were sacrificed and
brains removed, placed in ice cold PBS, and brain regions isolated by microdissection.
Samples were placed in PLC lysis buffer [12] supplemented with 1 mM sodium vanadate,
10 ug/ml leupeptin, 1 mM PMSF, 1 mM benzamidine, and 10 ug/ml aprotinin,
homogenized, then centrifuged at 14000 RPM at 4C to remove insoluble debris. Protein
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concentrations were determined by BCA protein assay (Pierce). For human tissues, a frozen
sample of frontal cortex was homogenized in PLC-lysis buffer with a polytron and then
incubated at 4C on a nutator for 2030 min to enhance solubilization of proteins. Samples
were then centrifuged and processed as above.
Antibodies and Western blot analysis
The ITSN1 antibody has been previously described [13]. Antibodies to neuron-specific
tubulin (Tuj1) and the hemagglutinin epitope (HA.11) are commercially available
(Covance). Western blot analyses were performed as described [12]. Differences in the
levels of ITSN1-S and ITSN1-L in patient samples were determined by densitometry using
ImageJ software (NIH) and normalized to the level of tubulin in each sample. The relative
expression of ITSN1-S and ITSN1-L was then compared between samples of various ages.
Locomotor activity

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A computerized AccuScan 12 Animal Activity Monitoring System (Columbus Instruments,


Columbus, OH) assisted by VERSAMAX software (AccuScan Instruments, Columbus, OH)
monitored locomotor activity in mice [14]. Each activity cage consisted of a Perspex box (20
20 20 cm) surrounded by horizontal and vertical infrared sensor beams. The stereotypic
time as a measure of the time the animal spent in stereotypic activity such as grooming or
head bobbing and the locomotor activity, including horizontal activity and center distance of
transgenic mice was recorded between 1:00 and 3:00 p.m. in the room housing the mice.

RESULTS

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ITSN1 mRNA levels are elevated in Down Syndrome [5]. However, little data exists on the
expression of ITSN1 proteins in human brains. Therefore, we examined the expression of
ITSN1 proteins in frontal cortex samples from normal and Down Syndrome cases (Table 1,
Supplemental Digital Content 2, http://links.lww.com/WNR/A148). The two major ITSN1
isoforms, ITSN1-S and ITSN1-L, were variably expressed in both normal and Down
Syndrome cases (see Figure 1, Supplemental Digital Content 3,
http://links.lww.com/WNR/A149). Comparison of ITSN1-S and ITSN1-L levels between
normal and Down Syndrome patients of varying ages indicated that ITSN1-S levels were
consistently elevated in Down Syndrome cases (p=0.023) (Figure 1A). Although ITSN1-L
levels were elevated in Down Syndrome vs control, this difference was not significant
(p=0.12). Since Down Syndrome patients above the age of 40 invariably develop an early
onset Alzheimers Disease-like neuropathology [2], we asked whether ITSN1 levels varied
as a function of age as well as genotype (control vs Down Syndrome) (Fig. 1B). For this
analysis, Down Syndrome and control cases were grouped as being under age 40 or age 40
and older as this is the typical age at which individuals with Down Syndrome have sufficient
neuropathology for a diagnosis of Alzheimers Disease. There did not appear to be a
significant variation in ITSN1 levels with age in control samples although Down Syndrome
cases under the age of 40 years exhibited elevated levels of both ITSN1-S and ITSN1-L
(p=0.021 and 0.035, respectively) (Fig. 1B). Interestingly, ITSN1 levels decreased in the
older vs younger Down Syndrome cases. To determine whether there was a correlation
between ITSN1 levels and Alzheimers Disease pathology in the Down Syndrome cases, we
stratified samples into control, Down Syndrome, and Down Syndrome with a concurrent
diagnosis of Alzheimers Disease (DS+AD) (Fig. 1C). Both ITSN1-S and ITSN1-L levels
are elevated in Down Syndrome patients (p=0.01 and 0.026) (Fig. 1C). However, ITSN1-S
and ITSN1-L levels are reduced in Down Syndrome+Alzheimers Disease cases compared
to Down Syndrome without Alzheimers Disease and these differences are statistically
significant (p=0.026 and 0.015, respectively). Using an independent set of brain samples
isolated for cases of similar age but harvested at varying times following death, we did not

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observe a systematic contribution of the time between tissue harvesting and death (postmortem interval, PMI) to the variability in ITSN1-S (Spearman r=0.61 p=0.14) or ITSN1L (Spearman r=0.90 p=0.85) levels across cases (see Figure 2, Supplemental Digital
Content 3, http://links.lww.com/WNR/A149).
These results indicate that ITSN1-S and ITSN1-L are upregulated in Down Syndrome
brains; however, the contribution of this overexpression to Down Syndrome is unknown. To
determine whether ITSN1 overexpression contributes to the sequelae of Down Syndrome,
we overexpressed ITSN1-S in the mouse brain. Several observations suggested that
overexpression of ITSN1-S may have a greater affect on brain function than ITSN1-L. First,
neurons normally express ITSN1-L, but not ITSN1-S [15,16]. Second, Down Syndrome
patients over 40 years of age develop an Alzheimers Disease-like neuropathology, and
ITSN1-S was shown to be specifically overexpressed in neurons from post-mortem
Alzheimers Disease brain samples [810]. Finally, overexpression of ITSN1-S increases
JNK activation and neurodegeneration [11].

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To determine if brain-specific overexpression of ITSN1-S results in observable phenotypes,


we generated mice that overexpress HA-tagged mouse ITSN1-S (HA-ITSN1-S) in the brain
by crossing tet-HAITSN1 mice with CaMKII-tTA mice [17]. The CaMKII-tTA drives
expression of a transgene from the Tet operon in neurons in forebrain structures with the
highest levels in the striatum [17,18]. Double transgenic CaMKII-tTA:tet-HAITSN1
(hereafter referred to as CaMKII-ITSN1) mice specifically express HA-ITSN1-S in
forebrain structures including the olfactory bulb, cerebral cortex, striatum/septum,
hippocampus, and thalamus/hypothalamus with highest levels in the striatum (Figure 2A).
Relatively low expression was detected in the olfactory bulb, thalamus, and midbrains. In
contrast, HA-ITSN1-S was not detected in the cerebellum or in the pons/medulla.
Endogenous ITSN1-S and ITSN1-L are ubiquitously expressed in the brains of single and
double transgenic mice, with ITSN1-L as the predominant isoform. However, ITSN1-S
expression was elevated relative to ITSN1-L levels in the CaMKII-ITSN1 samples (Fig. 2).
Comparison of total ITSN1 levels (endogenous and HA-ITSN1-S) in the striatums of single
(CaMKII and ITSN1) and double (CaMKII-ITSN1) transgenics reveals that the ratio of
ITSN1-S to ITSN1-L is significantly elevated in double compared to single transgenic
samples (Fig. 2B&C). These data indicate that HA-ITSN1-S constitutes a significant portion
of the total ITSN1 protein in this brain region. Further, the level of ITSN1-S overexpression
in the striatum of CaMKII-ITSN1 mice is comparable to the level of ITSN1-S
overexpression in the Down Syndrome brain samples, which indicates that this transgenic
line is biologically relevant for modeling the effects of ITSN1-S overexpression.

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To initially characterize the phenotypic consequences of ITSN1-S overexpression, we


measured the locomotor activity of single and double transgenics in an open field test. Male
CaMKII-ITSN1 mice exhibited an overall reduction in activity compared to the single
transgenic control mice (Figure 3). Double transgenics exhibited reduced horizontal activity
(CaMKII-ITSN1 vs CaMKII, p=0.031; CaMKII-ITSN1 vs ITSN, p=0.020; Fig. 3A),
exploratory behavior as measured by reduction in center distance (CaMKII-ITSN1 vs
CaMKII, p=0.007; CaMKII-ITSN1 vs ITSN1, p=0.025; Fig. 3B), stereotypy time (CaMKIIITSN1 vs CaMKII, p=0.037; CaMKII-ITSN1 vs ITSN1, p=0.005; Fig. 3C) and stereotypy
counts (CaMKII-ITSN1 vs CaMKII, p=0.029; CaMKII-ITSN1 vs ITSN1, p=0.009; Fig.
3D). Female double transgenics did not exhibit a statistically significant difference in
comparison to single transgenics (see Discussion).

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DISCUSSION
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ITSN1 is a critical regulator of endocytosis, synaptic transmission, and cell signaling [6]. Its
localization to HSA21 in the Down Syndrome Critical Region and elevated expression in
mouse models for Down Syndrome suggest that ITSN1 may contribute to the genesis and/or
progression of this disorder (reviewed in [6]). Although ITSN1 transcript levels are elevated
in the prosencephalon of Down Syndrome embryos [5], expression of ITSN1 proteins has
not been described. We demonstrate that ITSN1 levels are indeed elevated in frontal cortex
of Down Syndrome, with a clear distinction in expression of ITSN1 in younger (<40 yrs) vs
older (40) Down Syndrome cases. ITSN1-S and ITSN1-L isoforms were elevated in
younger Down Syndrome cases but in older Down Syndrome cases, levels of each isoform
were similar to controls. This age stratification of cases corresponds with the appearance of
an Alzheimers Disease-like neuropathology in Down Syndrome [2]. This correlation
suggests two possibilities: the decrease in ITSN1 levels may result from increased
neurodegeneration in older Down Syndrome patients leading to loss of ITSN1-expressing
cells; or ITSN1 overexpression may promote the loss of these neurons.

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To address the biological consequences of ITSN1 overexpression in vivo, we created a


novel transgenic mouse model in which ITSN1-S is specifically overexpressed in the
forebrain. Although neurons only express ITSN1-L (reviewed in [6]), our mouse model is
biologically relevant given the enhanced expression of ITSN1-S in Alzheimers Disease
brains [810] and the observed increases in ITSN1-S expression in Down Syndrome
patients. ITSN1-S overexpression resulted in reduced locomotor activity in male CaMKIIITSN1 mice. Although ITSN1-S overexpression did not result in statistically significant
effects in females, female mice expressing tTA alone showed reduced center distance (not
statistically significant, data not shown). Expression of tTA alone in the Tet system can have
a phenotypic effect [19,20]. However, since the differences in phenotypes between male
CaMKII-ITSN1 and control animals were statistically significant, we conclude that the
reduced activity in these mice is due to brain-specific ITSN1-S overexpression. The highest
level of expression of HA-ITSN1 was seen in the striatum, which regulates motor activity.
Thus, ITSN1-S may disrupt normal functioning of neurons in the striatum by interfering
with endocytic processes in these cells, altering the activity of various signaling pathways,
or a combination of both.

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Mouse models of Down Syndrome display abnormal behavior [21]. For example, Ts1Cje
mice are trisomic for 83 genes including ITSN1, and these mice are hypoactive [21,22].
However, this mouse model overexpresses many genes and therefore may not be
informative as to ITSN1s role in the abnormal behavioral phenotype. The consequences of
elevated ITSN1 levels in Down Syndrome patients remain unclear. ITSN1 overexpression
leads to endocytic defects, enhanced Ras activation, and increased receptor trafficking and
degradation (reviewed in [6]). Interestingly, ITSN1-S overexpression increases JNK MAPK
activation leading to enhanced neurodegeneration induced by polyglutamine expanded
proteins [11]. These data suggest that ITSN1 overexpression in Down Syndrome may
promote neurodegeneration through one or more of these pathways. This hypothesis is
consistent with the increase in ITSN1-S transcripts seen in Alzheimers Disease [810].
Thus, it is possible that ITSN1 cooperates with pathways to promote apoptosis of ITSN1overexpressing cells resulting in an age-dependent decrease of ITSN1 in Down Syndrome.
However, the role of ITSN1 in Down Syndrome may be more complex. ITSN1 regulates a
novel PI3K-AKT survival pathway in neurons [12] and may provide a compensatory
protective signal in Down Syndrome to counterbalance the negative effects of other
overexpressed HSA21 genes as suggested for several HSA21 genes in Down Syndrome
[23]. Additional studies using transgenic mice with targeted overexpression of ITSN1-L and
overexpression of both ITSN1-S and ITSN1-L in neurons may provide insight into the

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effects of ITSN1 in Down Syndrome. However, interpretation of such transgenic animals


may be complicated given the lack of combinatorial overexpression of HSA21 gene
products in single gene transgenics. Indeed, modeling the effects of three Drosophila
homologs of HSA21 genes (ITSN1, Synaptojanin, and RCAN1) reveals distinct phenotypes
in triple versus single or double transgenic flies [24].

Conclusion
Down Syndrome patients express elevated levels of ITSN1-S and ITSN1-L and exhibit a
precipitous decrease in ITSN1 protein in aged Down Syndrome cases coincident with the
development of Alzheimers Disease neuropathology consistent. Additionally, we have
shown that ITSN1-S overexpression in the mouse brain affects behavior.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

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Human tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University
of Maryland, Baltimore, MD. The role of the NICHD Brain and Tissue Bank is to distribute tissue, and therefore,
cannot endorse the studies performed or the interpretation of results. Funding for human tissue samples was from
UCI ADRC P50 AG16573, NICHD Contract No. N01-HD-4-3368 and NO1-HD-4-3383. M.K. was supported in
part by the Craig Fellowship, University of Illinois College of Medicine. This work was supported in part by
funding to G. P. from the NIH (MH 085999), to E.H. from the NIH (RO1 HD064993), and to J.P.O from the
Intramural Research Program of the NIH, the Foundation Jerome Lejeune, the Department of Defense (PR080428),
and the NIH (RO1 HL090651).

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Figure 1. Expression of ITSN1 in human frontal cortex samples

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(A) Relative expression of ITSN1-S and ITSN1-L was compared among all the samples.
Difference in ITSN1-S levels between normal and Down Syndrome was statistically
significant (p=0.023) using a Students t-test of the mean assuming unequal variance. (B)
Relative expression of ITSN1 isoforms upon stratification of samples by genotype and age.
Both control and Down Syndrome samples were segregated into below 40 or 40 yrs old and
older. Bars represent standard error. Statistically significant differences in ITSN1-S and
ITSN1-L (p=0.021 and p=0.035, respectively) in the younger Down Syndrome groupings
compared to younger controls are marked, *. These differences were lost in the older Down
Syndrome grouping. (C) Samples were stratified into control, Down Syndrome, Down
Syndrome with diagnosed Alzheimers Disease neuropathology (Down Syndrome
+Alzheimers Disease). Bars represent standard error. The difference in ITSN1-S and
ITSN1-L levels in Down Syndrome (p=0.01 and p=0.026, respectively) vs control samples
was statistically significant, *. As indicated in (C), these differences in ITSN1 levels were
lost in the older Down Syndrome+Alzheimers Disease grouping. This decrease in ITSN1
levels in Down Syndrome+Alzheimers Disease samples vs Down Syndrome was
statistically significant (**, p=0.026 for ITSN1-S; p=0.015 for ITSN1-L). Students t-test of
the mean assuming unequal variance was used to calculate p values in AC.

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Figure 2. Expression of HA-tagged ITSN1 in brain regions of CaMKII-ITSN1 mice

(A) Top panels, HA-ITSN1 expression in the different brain regions of transgenics. Single
transgenic control do not express HA-ITSN1. Middle panels, expression of endogenous
ITSN1. Bottom panels, tubulin as a loading control. (B) Expression of ITSN1-L and ITSN1S in the striatums of single transgenic controls and CaMKII-ITSN1 transgenic mice. ITSN1L predominates in the controls, but ITSN1-S predominates in the CaMKII-ITSN1. Tubulin
was used as a loading control. (C) The ratio of ITSN1-S to ITSN1-L levels in the striatums
was determined from blots in (B) using NIH ImageJ. The differences in ratios were
significant (p=0.022)

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Figure 3. Reduced activity of CaMKII-ITSN1 mice

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Results shown are for activity measurements during a 10 minute observation period. Male
CaMKII-ITSN1 mice show reduced (A) horizontal activity (measurement of the movement
of the animal in the horizontal plane of the open field chamber); (B) center distance (the
total distance an animal travels in center of open field which measures exploratory
behavior); (C) stereotypy time (time the animal spends in stereotypic activity such as
grooming or head bobbing); and (D) stereotypy counts (the number of beam breaks that
occur during stereotypic activity) compared to male single transgenic controls. Differences
between CaMKII-ITSN1 and single transgenics were statistically significant for all four
measures using Students t-test of the mean assuming unequal variance (P<0.05). Error bars
show SEM. The following numbers of animals and average ages were used for this analysis:
CaMKII-ITSN1: 9 M, 7.5+/0.71 (SD) months; CaMKII: 9 M, 8.4+/0.58 (SD) months;
ITSN1: 10 M, 7.1+/0.95 (SD) months.

Neuroreport. Author manuscript; available in PMC 2012 October 26.